The purpose of this study is to determine if human herpesvirus-6 (HHV-6), isolated from AIDS patients and associated with some B-cell lymphomas, contains transforming DNA sequences. To test this, HHV-6 genomic DNA and a few subclones were transfected into NIH 3T3 cells. Our data demonstrated that the NIH 3T3 cells can be repeatedly morphologically transformed by either the complete viral genome (HHV-6) or the subgenomic fragment in clone pZVH14. The frequency of focus formation was significantly above the background level. These focal lines demonstrated tumorigenic potential in both nude mice and immunocompetent mice. Moreover, tumorigenic transformants could also be derived by cotransfection of the DNA with a neomycin resistant marker. By histopathological examination, the tumors were identified to be undifferentiated fibrosarcomas. Southern blot hybridization revealed the presence of pZVH14 DNA sequences in the G418 selected transformed and tumor cell lines but not in the focus-derived cell lines. The G418 selected genomic DNA-induced lines were also positive for sequences related to another subclone pZVB70 of HHV-6. This clone was found to have weak transforming activity in a focus assay. Our failure to detect DNA sequences in the focus-derived lines could be due to the fact that 8.7 kb probe (pZVH14 insert) is too large to detect a small fragment (< 500bp) even if it exists in the transformed cells lines. pZVH14 DNA was not found to exist as extrachromosomal forms in these focal cell lines. These data suggest that HHV-6 DNA may be transiently integrated or a small piece of DNA is retained which could not be detected. Secondary transformants, derived by transfecting NIH 3T3 cells with genomic DNAs from the primary G418 selected transformed and tumor cell lines, exhibited much higher tumorigenic activity than the primary transformants. These data establish that HHV-6 contains transforming DNA sequences. The project is still active.